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ElisaBeginner 06-28-2013 06:05 AM

Elisa troubleshooting
Nice to be on this forum!
I would like to know something that I can't find on the internet no matter what I do:
1. LEt's suppose that you're doing an Elisa test, and positives lose color more and more, after adding the stop solution, until, after 3-4 minutes they are completely blank (transparent).Why does it happen?

ElisaBeginner 06-28-2013 06:07 AM

Re: Elisa troubleshooting
It's indirect Elisa, btw.

Zagami 06-28-2013 12:21 PM

Re: Elisa troubleshooting
Which substrate and stopping solution you use?

ElisaBeginner 07-01-2013 04:45 AM

Re: Elisa troubleshooting
TMB as substrate and 1N HCl as stopping solution.

Zagami 07-02-2013 05:54 AM

Re: Elisa troubleshooting
Stopping solutions of the TMB substrate
TMB has been shown to be a safe-sensitive substrate for the assay of horseradish peroxidase (HRP). Initially, in the presence of HRP and hydrogen peroxide, a one-electron oxidation product is formed. This compound, a cation free radical, is blue in colour with an adsorption maximum at 653 nm. Further reaction with HRP/H2O2 or acidification of the radical with acid yields the diimine terminal oxidation product yellow with an absorbance peak at 450 nm. The extinction coefficient of the radical (Abs653 nm = 3.9 x 10^4 mol^-1 cm^-1) and diimine (Abs450 nm = 5.9 x 10^4 mol^-1 cm^-1) provide a remarkably sensitive system for the assay of HRP and HRP labelled probes. Several acid solutions may be used to stop the TMB reaction. A recommended acid stop solution is 1 N H2SO4 or a mixture of HCl and H2SO4 (final concentration equals 1 N HCl and 3 N H2SO4) or with sodium fluoride. In endpoint assays, the substrate reaction can be stopped with 1 N HCl or 0.6 N sulfuric acid. The intensity of the acidified reaction product may change over time. For best results, specially when used 1 N HCl, to read immediately after adding acid stop solution. We have observed that 0.5 mol/L-1 hydrochloric acid (adding 83 ml of 6 mol/L AR HCl to 917 ml of reagent grade water must contain less than 10-7 mol/L^-1 of iron or copper salts otherwise unreacted TMB will be converted non-enzymatically to the diimine) converts the blue radical to the yellow diimine and the yellow colour is stable for at least 30 minutes after the addition of acid. Same result has been obtained employing a 0.3 - 1 M phosphoric acid (H3PO4) solution provides the most stable endpoint color.

JonesMiller 07-22-2013 07:29 AM

Re: Elisa troubleshooting
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